ORIGINAL ARTICLE

http://dx.doi.org/10.1590/S1678-9946201860037

Prospective cohort studies to evaluate the safety and immunogenicity of the 2013, 2014, and 2015 seasonal influenza produced by

Gabriella Mondini1, Patricia Emilia Braga1, Marta Heloisa Lopes2, Ana Marli Christovam Sartori2, Karina Takesaki Miyaji2, Vanessa Infante2, Bruno Azevedo Randi2, Maria do Carmo Sampaio Tavares Timenetsky3, Juliana Caires de Oliveira Achili Ferreira4, Neusa Keico Sakita4, Alexander Roberto Precioso1,5

ABSTRACT

Annual vaccination is the most effective way to prevent seasonal influenza illness. Instituto Butantan (IB) performed clinical studies with its 2013, 2014 and 2015 seasonal trivalent influenza vaccines (inactivated split-virion). Prospective cohort studies were carried out to describe the safety and immunogenicity of Instituto Butantan influenza vaccines, in healthy adults and elderly, from 2013 to 2015. Immediately after the informed consent was signed, participants underwent blood collection followed by vaccination. On study days 1, 2 and 3 post-vaccination participants were contacted by the staff to evaluate the occurrence of solicited (local and systemic) and non-solicited adverse reactions. On study day 21 (+7) subjects returned to the clinical site for final safety assessments and blood collection to

1Instituto Butantan, Divisão de Ensaios evaluate post-vaccination immunogenicity. The immunogenicity analyses were performed Clínicos e Farmacovigilância, São Paulo, by means of hemagglutination inhibition (HI) assay. The immunogenicity endpoints were: São Paulo, seroprotection (SPR) and seroconversion (SCR) rates and the geometric mean HI antibody titer ratio (GMTR). The 2013 study was conducted at the Centro de Referência para 2Universidade de São Paulo, Faculdade de Imunobiológicos Especiais (CRIE) and at the Centro de Pesquisa Clínica do Instituto da Medicina, Hospital das Clínicas, Centro de Referência para Imunobiológicos Especiais Criança, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (CRIE), São Paulo, São Paulo, Brazil while the 2014 and 2015 studies were conducted at CRIE. The composition followed the WHO recommendation for the Southern hemisphere seasonal influenza vaccine. Forty- 3Instituto Adolfo Lutz, Centro de Virologia, seven healthy adults and 13 elderly participated in the 2013 study, 60 healthy adults and 60 São Paulo, São Paulo, Brazil elderly in the 2014 study, and 62 healthy adults and 57 elderly in the 2015 study. In the 2013, 4Universidade de São Paulo, Faculdade de 2014 and 2015 studies, pain was the most frequent local adverse reaction and headache the Medicina, Hospital das Clínicas, Instituto da most frequent systemic adverse reaction. All observed adverse reactions were classified as Criança, São Paulo, São Paulo, Brazil mild or moderate and none as severe. SPR >70% and SPR >60% were observed in adults 5Universidade de São Paulo, Faculdade de and elderly, respectively, for the three vaccine viruses, in the 2013, 2014 and 2015 studies. Medicina, Departamento de Pediatria, São SCR >40% was observed in adults, for the three vaccine viruses, only in the 2014 study and Paulo, São Paulo, Brazil SCR >30% was observed in the elderly, for the three vaccine viruses, only in the 2013 and

Correspondence to: Alexander Roberto 2014 studies. GMTR >2.5 among adults, for the three vaccine viruses was only observed in Precioso the 2013 study and GMTR >2.0 was observed among elderly, for the three vaccine viruses, Instituto Butantan, Divisão de Ensaios in the 2013, 2014 and 2015 studies. The 2013, 2014 and 2015 seasonal influenza vaccines Clínicos e Farmacovigilância, Avenida Vital produced by Instituto Butantan were safe and immunogenic according to the immunogenicity Brasil, 1500, CEP 05503-900, São Paulo, criteria defined by the European Agency (EMA). SP, Brazil

E-mail: [email protected] KEYWORDS: Seasonal influenza vaccine. Vaccine immunogenicity. Vaccine safety. Cohort studies. Adults. Elderly. Received: 6 April 2018

Accepted: 2 July 2018

Rev Inst Med Trop São Paulo. 2018;60:e37 This is an open-access article distributed under the Page 1 of 11 terms of the Creative Commons Attribution License. Mondini et al.

INTRODUCTION at the Centro de Pesquisa Clínica do Instituto da Criança (ICR) both from the Hospital das Clínicas da Faculdade Influenza or Flu is a viral disease of high transmissibility de Medicina da Universidade de São Paulo (HCFMUSP). that affects the respiratory tract of people at any age and However, the 2014 and 2015 studies were conducted has global distribution. According to the World Health exclusively at CRIE. The three studies were performed Organization (WHO), this disease results in 3 to 5 million within the period of the Brazilian national immunization of serious cases and 250,000 to 500,000 deaths, annually1. campaign for influenza, and all were approved by the Ethics People at increased risk to develop complications Committee of the Faculdade de Medicina da Universidade associated with influenza virus include adults older than de São Paulo and were registered at ClinicalTrials.gov 65 years, pregnant women, immunocompromised patients, (2013 study: NCT02819115, 2014 study: NCT02819180, people with chronic underlying medical conditions, children and 2015 study: NCT 02313740). The three studies under 5 years of age and health care professionals2,3. were conducted in accordance with the principles of Annual vaccination is recommended by WHO as the most the Declaration of Helsinki, Good Clinical Practices6, effective way of preventing seasonal influenza. In Brazil, Resolution 466/2012 of the Brazilian National Health the National Immunization Program (NIP) of the Ministry Council (CNS) on ethics in human research and financially of Health has carried out annual national influenza vaccine sponsored by Butantan Foundation. campaigns since 1999. In the first national immunization campaign, the NIP recommended vaccination only for adults Subjects and study procedures over 65 years of age, however, the recommendation has progressively been expanded to other target groups such as Healthy, male and non-pregnant female between 18 children 6 months to 5 years, adults over 60 years, pregnant and 59 years of age, and elderly over 60 years of age were women, women in the post-partum period (up to 45 days), eligible for enrollment and the participants were selected healthcare professionals, indigenous people, people from 12 based on inclusion and exclusion criteria (described to 21 years under socio-educational programs (adolescents below). All participants provided written informed consent being followed in freedom or not for different kind of and were invited to participate at the time they presented conflicts with law), people deprived of freedom, employees of spontaneously at the clinic where the study was taking place. the prison system, people with chronic and/or other medical These predefined age groups followed the recommendation conditions4. In Brazil, the NIP has provided the inactivated by the European Medicines Agency (EMA) for studies of split-virion seasonal trivalent influenza vaccine (inactivated seasonal influenza vaccines7,8. split-virion) free of charge to the target populations5. Inclusion criteria: Instituto Butantan (IB) is a public Brazilian biomedical 1. Healthy adults, male or female aged 18 to 59 or elderly research manufacturer center affiliated to the Sao Paulo aged 60 years and above; State Secretary of Health. Currently, it is one of the main 2. To be available to participate in the study throughout public producers of vaccines, , and antitoxins its duration (approximately 21 days); in Latin America. 3. To have medical indication to be vaccinated against From 2013 to 2015, the IB’s Division of Clinical Trials influenza; and Pharmacovigilance performed prospective cohort studies 4. To demonstrate intention to participate in the study, as to evaluate the safety and immunogenicity of the 2013, 2014, documented by the signature in the study’s informed and 2015 seasonal trivalent influenza vaccines (inactivated consent form (ICF). split-virion) in health adults and people over 60 years of Exclusion criteria: age. Here, we describe the results of those studies, which 1. Evidence of active neurological, cardiac, pulmonary, were requested by the Brazilian regulatory health authority hepatic or renal disease as clinical history and/or (Agência Nacional de Vigilância Sanitária – ANVISA). physical examination (except hypertension under control among the elderly); MATERIAL AND METHODS 2. Compromised immune system diseases including: HIV, diabetes mellitus, cancer (except basal cell carcinoma) Study designs and autoimmune diseases; 3. Behavioral, cognitive or psychiatric disease that in the In 2013, 2014, and 2015 we performed prospective opinion of the principal investigator or his representative cohort studies. The 2013 study was conducted at the Centro physician, affects the participant ability to understand de Referência de Imunobiológicos Especiais (CRIE) and and cooperate with all study protocol requirements;

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4. Alcohol or drugs abuse in the past 12 months that were oriented to take the axillary temperature and record has caused medical, professional or family problems, it in the participant card for the following three days. indicated by clinical history; Fever was defined as an axillary temperature≥ 37.8 ºC. 5. Known systemic hypersensitivity to eggs or to any Participants were also contacted by telephone three days component of the vaccine; after vaccination to check the occurrence of any unsolicited 6. History of severe adverse reaction after previous and solicited local (pain, bruising, redness, and swelling), administration of an Influenza vaccine within 6 weeks and systemic (fever, chills, myalgia, fatigue, and headache) following vaccination; adverse reactions (AR). On day 21 (+7) post-vaccination 7. History of Guillain-Barré Syndrome or other participants returned to the research clinic for the post- demyelinating disease; vaccination immunogenicity analysis and to check the 8. Diagnosis of asthma with a history of hospitalization in participant card. the last six months due to the illness; 9. Suspected or confirmed fever in the 3 days prior to Endpoints vaccination or axillary temperature greater than 37.8 °C on the day of vaccination; Safety endpoint 10. Use of corticosteroids (except topical or nasal) or The primary safety endpoints were solicited and other immunosuppressive drugs within 42 days before unsolicited local and systemic adverse reactions reported the study initiation/baseline. It will be considered by the participants until day 3 after vaccination. Adverse immunosuppressive dose of corticosteroids the equivalent reactions were defined as adverse events that had reasonable to a dose ≥ 10 mg of prednisone per day for over 14 days; causal relationship to vaccination, as defined by the adapted 11. Impaired coagulation due to chronic disease or due to classification of “Uppsala Monitoring Centre” of the World the use anticoagulant medication (warfarin or heparin) Health Organization9. The intensity of adverse reactions in the 7 days preceding vaccination; was classified as grade one to four according to the Toxicity 12. Have received live virus vaccine within 28 days or killed Grading Scale for Healthy Adult and Adolescent Volunteers virus vaccine in the last 14 days prior to vaccination, Enrolled in Preventive Vaccine Clinical Trials of the US or having a scheduled immunization during the first Food and Drug Administration (FDA)10. 21 days after vaccination; 13. Have received influenza vaccine in the past 6 months; Immunogenicity endpoints 14. History of asplenia; The three co-primary immunogenicity endpoints were 15. Have received blood products in the past 6 months, the proportion of participants that presented seroconversion including transfusions or immunoglobulin, or scheduled (pre-vaccination hemagglutination inhibition (HI) administration of blood products or immunoglobulin for antibody titer <1:10 and post-vaccination HI antibody the first 21 days after vaccination; titer ≥1:40, or pre-vaccination HI antibody titer <1:10 and 16. have a counter indication for Influenza vaccination, a post-vaccination increase by a factor of four or more), including allergy to egg proteins; the proportion of participants with seroprotection (post- 17. Use of any investigational product within 42 days before vaccination HI antibody titer ≥1:40), and the factor increase vaccination; in the geometric mean titer, all measured before and 21 days 18. Any other condition that might put in risk the safety/ after each vaccination. rights of a potential participant or hurdle his/her The immunological criteria for evaluation of influenza compliance with this protocol in the investigator’s vaccines were based on the Note for Guidance on opinion or his representative physician. Harmonization of Requirements for Influenza Vaccines of “The European Medicines Agency (EMA)”7. According Procedures to EMA a seasonal influenza vaccine is considered immunogenic when one of the immunogenicity endpoints is Participants were included in the study soon after demonstrated accordingly with the age7. The immunological their inclusion, exclusion criteria were checked and the criteria for the evaluation of seasonal influenza vaccines are informed consent signed. A blood sample of 5 ml was taken described in Table 1. before vaccination and on day 21 (+7) post-vaccination (for immunogenicity analysis). Participants received one Laboratory assays dose of the vaccine intramuscularly and were observed for 30 min at the research clinical site. Before leaving, they Anti-influenza antibody response was measured by the

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Table 1 - Immunological criteria for the evaluation of seasonal measures of central tendency and dispersion (age). influenza vaccines For each study year sex, ethnicity and number of participants with adverse reactions were compared between Parameters – one or more of the 18 - 60 > 60 groups using Chi-square test or Fisher’s exact test. following: years years The safety analysis was described to all participants Seroconversion rate (SCR) >40% >30% using the percentages of adverse reactions (AR), solicited Geometric mean titer ratio (GMTR) >2.5 >2.0 and non-solicited, by age group and study year. These Seroprotection rate (SPR) >70% >60% frequencies were compared by the Chi-square test or the Fisher’s exact test. hemagglutination-inhibition assay, according to standard The immunogenicity endpoints were analyzed for each method at Adolfo Lutz Institute (IAL) (Sao Paulo, Brazil). age group, by influenza vaccine strain and study year. In Titers were tested at an initial dilution of 1:10, and at a final the immunogenicity analysis, only participants with pre and dilution of 1:2,560. For the purposes of calculation, negative post-vaccination blood samples available were included. titers were assigned a value of 1:5 and those with titers The geometric mean HI antibody titer, pre- and post- above 1:2,560 had assigned a value of 1:2,560. Samples vaccination, and the GMTR were calculated with respective were tested in duplicate, and the geometric mean value was 95% confidence intervals (CI). The SCR, SPR and the used in the analyses7,11. proportion of participants with pre-vaccination HI antibody titer ≥1:40 were also calculated with the respective 95% CI. Vaccines To each age group, by study year and influenza vaccine strain the percentages of SCR, SPR and the GMTR were The composition of seasonal trivalent influenza calculated stratified by pre-vaccination HI antibody titer vaccines (inactivated split-virion) produced by IB and results (<1:40 or ≥1:40). The Mann-Whitney test was used evaluated in this study followed the WHO composition to assess the significance of differences in GMTR between recommendation for the Southern Hemisphere. Their strata. SCRs were compared using the the Fisher’s exact test. compositions were: 2013 (A//7/2009 (H1N1) Statistical analyses were performed using a significance pdm09-like virus; A/Victoria/361/2011 (H3N2)-like virus level of 5%. The software Stata 13.0 (StataCorp LP, College B/Wisconsin/1/2010-like virus); 2014 (A/California/7/2009 Station, Texas USA) was used in the analyses. (H1N1)pdm09-like virus, A/Texas/50/2012 (H3N2)- like virus, B/Massachusetts/2/2012-like virus), and RESULTS 2015 (A/California/7/2009 (H1N1)pdm09-like virus; A/Switzerland/9715293/2013 (H3N2)-like virus; Study participants B/Phuket/3073/2013-like virus. The vaccines were formulated in a 10-dose vials (0.5mL per dose) and stored The 2013, 2014, and 2015 studies were performed at 2-8 ºC until used. from June 4 to July 27th, April 22nd to June, and May 23rd to June 23rd, respectively. The participant demographic Sample size characteristics are described in Table 2. The total number of participants included in the safety and immunogenicity The planned sample size for the 2013 study was 100 analysis is described in Figure 1. participants (50 adults and 50 elderly) and for the 2014 and In 2013, 63 volunteers agreed to participate in the study 2015 studies, 120 participants (60 adults and 60 elderly). and signed the informed consent (63% of the planned These sample sizes were accepted by ANVISA and followed sample size): 50 adults and 13 elderly. However, only 47 the EMA7 recommendation to demonstrate safety and (94%) adults were included in the safety analysis, since immunogenicity of seasonal influenza vaccine. two of them had been vaccinated with an influenza vaccine not produced by IB and one participant presented with Statistical analysis high blood pressure on the vaccination day and, therefore, was not vaccinated. The median age of the adults was 33.3 In this study, we performed a descriptive statistical years, and the elderly 67.8 years. Only 13 (26%) of the analysis stratified by participants age groups: adults up elderly participants agreed to participate in the study and to 59 years and elderly above 60 years. The demographic signed the informed consent and all were included in the characteristics of the participants were described by safety analysis. percentage (gender and ethnicity) and by estimated In 2014, 120 volunteers agreed to participate in the study

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Table 2 - Demographic characteristics of participants according to the vaccination study year and age group

2013 Study 2014 Study 2015 Study n=60 n=120 n=119 VARIABLES Adults Elderly Adults Elderly Adults Elderly p* p** p** (n=47) (n=13) (n=60) (n=60) (n=62) (n=57)

Age (P50 (P25– P75)) 33.3 67.8 NA 30.1 66.9 NA 31.5 68.3 NA (25.3–44.8) (62.3–70.0) (27.2–42.4) (62.9–73.1) (27.7– 42.0) (65.5–75.0) Sex (n (%)) 0.045 0.845 0.725 Female 29 (61.7) 12 (92.3) 41 (68.3) 40 (66.7) 40 (64.5) 35 (61.4) Male 18 (38.3) 1 (7.7) 19 (31.7) 20 (33.3) 22 (35.5) 22 (38.6) Ethnicity (n (%)) 0.159*** 0.001*** 0.977*** White 38 (80.8) 8 (61.5) 42 (70.0) 24 (40.0) 48 (77.4) 44 (77.2) Black 2 (4.3) - 4 (6.7) 2 (3.3) 1 (1.6) 2 (3.5) Multirracial 3 (6.4) 1 (7.7) 1 (1.7) 1 (1.7) 9 (14.5) 2 (3.5) Asian 4 (8.5) 2 (15.4) 4 (6.6) 2 (3.3) 4 (6.5) 8 (14.0) Other - 2 (15.4) 9 (15.0) 31 (51.7) - 1 (1.8) NA: Not Applicable; (*) Fisher´s exact test; (**) Chi-square test; (***) white x others

and signed the informed consent. All of them were included in the safety analysis. The median age of the adults was 30.1 years and the elderly 66.9 years. In 2015, 120 agreed to participate in the study and signed the informed consent, however, only 119 (99.2%) were included in the safety analysis, 62 adults and 57 elderly. One participant was excluded from safety analysis because he was vaccinated with an influenza vaccine not produced by IB. The median age of the adults was 31.5 and the elderly 68.3 years.

Safety data

Table 3 shows the descriptive statistical analysis of the adverse reactions according to study year and age group. Table 4 shows the descriptive statistical analysis of the local and systemic solicited and non-solicited adverse reactions, by study year and age group. In the 2013 study, the total number of adult and elderly participants with ARs was 24 (51%) and four (30.8%), respectively. There was no significant statistical difference in the incidence of ARs between adults and elderly (p=0,226). The total number of ARs was 40, 34 (85%) in adults and six (15%) in the elderly. The mean number of ARs was 1.4 per adult and 1.5 per elderly. Thirty-two (94.1%) out of 34 ARs observed in adults were classified as grade I (mild) and two (5.9%) as grade II (moderate). All adverse reactions reported by the elderly were classified as grade I (mild). The mean interval between the time of vaccination and the onset of ARs in adults was 1.2 days and in the elderly 0.3 days. The mean duration of the ARs among adults was one day and among the elderly 6.5 days. Pain Figure 1 - Flowchart with the number of participants included at the injection site and headache were the most frequent and analyzed by the study endpoints, year and age group solicited, local and systemic ARs in both adults and elderly.

Rev Inst Med Trop São Paulo. 2018;60:e37 Page 5 of 11 Mondini et al.

Table 3 - Statistical analysis of the adverse reactions according to study year and age group

2013 Study 2014 Study 2015 Study n=60 n=120 n=119 VARIABLES Adults Elderly Adults Elderly Adults Elderly p* p* p* (n=47) (n=13) (n=60) (n=60) (n=62) (n=57) N of participants with 24 (51.0%) 4 (30.8%) p=0.226* 41 (68.3%) 16 (26.7%) p<0.001** 33 (53.2%) 16 (28.1%) p=0.005** ARs N of ARs 34 6 85 25 61 34 N of ARs per participant*** mean (sd) 1.4 (0.7) 1.5 (0.6) 2.1 (1.4) 1.6 (1.2) 1.8 (1.5) 2.1 (1.8) median (min-max) 1 (1 - 3) 1.5 (1 - 2) 1 (1 - 5) 1 (1 - 5) 1 (1 - 8) 1.5 (1 - 7) ARs grade/intensity 1 32 (94.1%) 6 (100%) 85 (100%) 25 (100%) 49 (80.3%) 24 (70.6%) 2 2 (5.9%) - - - 12 (19.7%) 10 (29.4%) AR onset (days) from vaccination mean (sd) 1.2 (0.9) 0.3 (0.8) 0.4 (0.6) 0.6 (0.9) 0.8 (0.8) 0.4 (0.8) median (min-max) 1 (0 - 3) 0 (0 - 2) 0 (0 - 3) 0 (0 - 3) 1 (0 - 3) 0 (0 -3) ARs duration (days) mean (sd) 1.0 (2.2) + 6.5 (9.3) ++ 1.9 (1.2) 2.0 (1.2) 1.4 (4.4) 1.1 (1.0) median (min-max) 0 (0 - 12)+ 3 (0 - 20)++ 2 (0 - 7) 2 (0 - 6) 1 (0 - 34) 1 (0 - 3) Abbreviation: AR, adverse reaction. (*) Fisher´s exact test; (**) Chi-square test; (***) Among those who presented AR. (+) 4 ARs with missing data, (++) 2 ARs with missing data.

Table 4 - Local and systemic solicited and non-solicited adverse reactions according to the study year and age group

2013 Study 2014 Study 2015 Study Adverse Reactions Adults Elderly Adults Elderly Adults Elderly p* p** p** (n=47) (n=13) (n=60) (n=60) (n=62) (n=57) Solicited reactions Local Pain 15 (31.9) 2 (15.4) 0.314 40 (66.7) 12 (20.0) <0.001 29 (46.8) 13 (22.8) 0.007 Erythema 1 (2.1) - 0.596 - - - 1 (1.6) 2 (3.5) 0.606 Swelling - - - 2 (3.3) - 0.496 4 (6.5) 2 (3.5) 0.681 Induration 1 (2.1) - 0.296 6 (10.0) 3 (5.0) 0.491 3 (4.8) 1 (1.8) 0.620 Ecchymosis - 1 (7.7) 0.217 2 (3.3) 1 (1.7) >0.999 1 (1.6) 1 (1.8) >0.999 Systemic Fever 3 (6.4) - 0.350 1 (1.7) 2 (3.3) >0.999 3 (4.8) 3 (5.3) >0.999 Chills 1 (2.1) - 0.596 5 (8.3) - 0.057 1 (1.6) 3 (5.3) 0.348 Headache 4 (8.5) 1 (7.7) 0.925 11 (18.3) 3 (5.0) 0.086 7 (11.3) 4 (7.0) 0.533 Fatigue - - - 8 (13.3) 2 (3.3) 0.047 2 (3.2) 1 (1.8) >0.999 Myalgia - - - 10 (16.7) 2 (3.3) 0.029 5 (8.1) 2 (3.5) 0.442 Unsolicited reactions Sore throat 2 (4.2) - >0.999 ------Cough 3 (6.4) - >0.999 ------Rhinorrhoea 3 (6.4) 1 (7.7) >0.999 ------Dry mouth ------1 (1.6) - >0.999 Arthralgia ------1 (1.6) - >0.999 Insonmnia ------1 (1.6) - - Nausea ------1 (1.8) 0.479 Pruritus 1 (2.1) 1 (7.7) 0.389 ------Local Pruritus ------2 (3.2) 1 (1.8) >0.999 (*) Fisher´s exact test; (**) Chi-square test

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There was no significant statistical difference with respect and influenza B viruses (Table 5). In adults SPR >70%, the incidence of local and systemic solicited ARs between SCR >40% and GMTR >2.5 were described for the three adults and elderly. vaccine viruses (Table 5). Adults with pre-immunization HI In the 2014 study, the total number of adult and elderly antibodies titers <1:40 had significantly higher GMTRs for participants with ARs was 41 (68.3%) and 16 (26.7%), the three vaccine antigens when compared to those with pre- respectively. The incidence of ARs was statistically immunization HI antibodies titers ≥1:40 (H1N1: p<0.001; significant higher among adults when compared to elderly H3N2: p=0.023; B: p<0.001) (Table 6). (p<0.001). The total number of ARs was 110, 85 (77.3%) The SCR for H1N1 vaccine virus was also significantly in adults and 25 (22.7%) in the elderly. The mean number higher among the adults with pre-immunization HI of ARs was 2.1 per adult and 1.6 per elderly. All the ARs antibodies titers <1:40 when compared to those with observed in adults and elderly were classified as grade I pre-immunization HI antibodies titers ≥1:40 (p<0.001). (mild). The mean interval between the time of vaccination However, there was no significant statistical difference in and the onset of ARs in adults was 0.4 days and in the elderly the SCR for H3N2 (p=0.454) and for influenza B (p=0.005) 0.6 days. The mean duration of the ARs among adults was vaccine viruses regardless the pre-immunization HI 1.9 day and among elderly 2.0 days. Pain at the injection antibodies titers (Table 6). In the elderly, SPR >60%, SCR site and headache were the most frequent solicited, local >30% and GMTR >2.0 were described for the three vaccine and systemic ARs in both adults and elderly. Pain at the viruses (Table 5). The elderly with pre-immunization HI injection site (p<0.001), fatigue (p<0.047) and myalgia antibodies titers <1:40 had significantly higher GMTRs (p<0.029) were statistically more frequent in adults when for H1N1 and H3N2 vaccine viruses when compared to compared to elderly. those with pre-immunization HI antibodies titers ≥1:40 In the 2015 study, the total number of adult and elderly (p<0.009 and p=0.011, respectively) (Table 6). However, participants was 33 (53.2%) and 16 (28.1%), respectively. there was no significant statistical difference in the GMTR The incidence of ARs was statistically significant higher for influenza B vaccine virus (p=0.337) regardless of the among adults when compared to elderly (p=0.005). The pre-immunization HI antibodies titers (Table 6). total number of ARs was 95, being 61 (64.2%) in adults and The SCR for the H3N2 vaccine virus was also 34 (35.8%) in the elderly. The mean number of ARs was statistically higher among the elderly with pre-immunization 1.8 per adult and 2.1 per elderly. Forty-nine (80.3%) out of HI antibodies titers <1:40 when compared to those with 61 ARs observed in adults were classified as grade I (mild) pre-immunization HI antibodies titers ≥1:40 (p=0.007). and 12 (19.7%) as grade II (moderate). Twenty-four (70.6%) However, there was no statistically significant difference in out of 34 ARs observed in the elderly were classified as the SCR for the H1N1 (p=0.192) and influenza B p=( 0.530) grade I (mild) and 10 (29.4%) as grade II (moderate). The vaccine viruses regardless of the pre-immunization HI mean interval between the time of vaccination and the antibodies titers (Table 6). onset of ARs in adults was 0.8 days and in the elderly 0.4 In the 2014 study, 115 (95.8%) out of 120 participants days. The mean duration of the ARs among adults was 1.4 completed their pre-vaccination and post-vaccination blood day and among the elderly 1.1 days. Pain at the injection sample collection and, therefore, were included in the site and headache were the most frequent solicited, local immunogenicity analysis: 57 adults and 58 elderly. Before and systemic ARs in both adults and elderly. Pain at the immunization, 84.2% of the adults had HI antibodies titers injection site (p<0.007) was statistically more frequent in ≥1:40 for H1N1 virus, 77.2% for H3N2 virus, and 87.7% adults when compared to elderly. for influenza B virus; among the elderly, 56.9% had HI antibodies titers ≥1:40 for H1N1 virus, 81% for H3N2 virus, Immunogenicity data and 74.1% for influenza B virus (Table 5). In adults SPR >70% and SCR <40% were described for the three vaccine Tables 5 and 6 show the immunogenicity results of 2013, viruses. GMTR >2.5 was only found for H1N1 and H3N2 2014 and 2015 studies. vaccine viruses (Table 5). Adults with pre-immunization HI In the 2013 study, only 55 (91.7%) out of 60 participants antibodies titers <1:40 had significantly higher GMTRs for had pre and post-vaccination blood samples collected, the three vaccine viruses when compared to those with pre therefore were included in the immunogenicity analysis: immunization HI antibodies titers ≥1:40 (H1N1 p=0.011; 42 adults and 13 elderly. Before immunization, 50% of H3N2 p<0.001; influenza Bp <0.001) (Table 6). the adults had HI antibodies titers ≥1:40 for H1N1, H3N2, The SCR for H3N2 and influenza B vaccine viruses and influenza B viruses; among elderly, 53.8% had HI were also significantly higher among adults with pre antibodies titers ≥1:40 for H1N1 virus and 30.8% for H3N2 immunization HI antibodies titers <1:40 when compared

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Table 5 - Pre- and post-vaccination hemagglutination-inhibition antibodies titers, geometric mean titer ratio, seroprotection and seroconversion rates distributed by age group and study year

2013 Study 2014 Study 2015 Study RESULTS Adults (n=42) Elderly (n=13) Adults (n=57) Elderly (n=58) Adults (n=59) Elderly (n=56) (CI 95%) (CI 95%) (CI 95%) Pre-vaccination HI ≥1:40 H1N1 50.0% (34.2 – 65.8) 53.8% (25.1 – 80.8) 84.2% (72.1 – 92.5) 56.9% (43.2 – 69.8) 78.0% (65.3 – 87.7) 39.3% (26.5 – 53.2) H3N2 50.0% (34.2 – 65.8) 30.8% (9.1 – 61.4) 77.2% (64.2 – 87.3) 81.0% (68.6 – 90.1) 57.6% (44.1 – 70.4) 48.2% (34.7 – 62.0) B 50.0% (34.2 – 65.8) 30.8% (9.1 – 61.4) 87.7% (76.3 – 94.9) 74.1% (61.0 – 84.7) 88.1% (77.1 – 95.1) 62.5% (48.5 – 75.1) GMT H1N1 31.2 (19.3 – 50.5) 32.3 (13.4 – 77.8) 72.6 (54.2 – 97.2) 31.5 (23.3 – 42.6) 69.5 (53.3 – 90.6) 23.8 (17.9 – 31.5) H3N2 24.0 (16.1 – 35.8) 23.5 (10.5 – 52.5) 74.4 (51.4 – 107.6) 78.1 (56.5 – 108.0) 33.1 (23.9 – 45.9) 29.0 (22.1 – 38.0) B 24.8 (17.3 – 35.5) 18.0 (10.8 – 29.9) 180.7 (125.3 – 260.6) 57.9 (44.4 – 75.7) 81.9 (64.8 – 103.6) 38.5 (30.1 – 49.4) Post-vaccination GMT H1N1 167.0 (119.0 – 234.4) 272.7 (167.3 – 444.4) 187.4 (145.9 – 240.8) 123.0 (94.0 – 160.9) 134.1 (106.2 – 169.5) 59.4 (45.0 – 78.5) H3N2 230.0 (153.8 – 344.1) 198.0 (99.1 – 395.7) 209.1 (165.3 – 264.5) 231.7 (182.8 – 293.7) 182.1 (140.9 – 235.3) 178.9 (128.6 – 248.7) B 89.8 (67.2 – 120.0) 80.0 (43.2 – 148.2) 408.1 (305.5 – 545.3) 161.9 (125.6 – 208.8) 143.9 (115.0 – 180.2) 100.0 (78.4 – 127.5) GMTR H1N1 5.4 (3.52 – 8.1) 8.4 (3.8 – 19.0) 2.6 (2.2 – 3.1) 3.9 (3.1 – 5.0) 1.9 (1.6 – 2.4) 2.5 (2.1 – 3.0) H3N2 9.6 (6.5 – 14.1) 8.4 (3.4 – 21.0) 2.8 (2.1 – 3.7) 3.0 (2.2 – 3.9) 5.5 (4.1 – 7.3) 6.2 (4.7 – 8.2) B 3.6 (2.7 – 4.8) 4.5 (2.4 – 8.2) 2.3 (1.8 – 2.8) 2.8 (2.2 – 3.5) 1.8 (1.5 – 2.1) 2.6 (2.1 – 3.2) SCR H1N1 57.1% (41.0 – 72.3) 76.9% (46.2 – 95.0) 38.6% (26.0 – 52.4) 48.3% (35.0 – 61.8) 15.3% (7.2 – 27.0) 25.0% (14.4 – 38.4) H3N2 78.6% (63.2 – 89.7) 61.5% (31.6 – 86.1) 31.6% (19.9 – 45.2) 37.9% (25.5 – 51.6) 72.9% (59.7 – 83.6) 67.9% (54.0 – 79.7) B 52.4% (36.4 – 68.0) 69.2% (38.6 – 90.9) 17.5% (8.7 – 29.9) 37.9% (25.5 – 51.6) 13.6% (6.0 – 25.0) 39.3% (26.5 – 53.2) SPR H1N1 95.2% (83.8 – 99.4) 100% (75.3 – 100) 96.5% (87.9 – 99.6) 93.1% (83.3 – 98.1) 94.9% (85.9 – 98.9) 66.1% (52.2 – 78.2) H3N2 95.2% (83.8 – 99.4) 92.3% (64.0 – 99.8) 98.2% (90.6 – 100) 98.3% (90.8 – 100) 98.3% (90.9 – 100) 87.5% (75.9 – 94.8) B 90.5% (77.4 – 97.3) 92.3% (64.0 – 99.8) 100% (93.7 – 100) 98.3% (90.8 – 100) 96.6% (88.3 – 99.6) 87.5% (75.9 – 94.8) Abbreviations: GMT, Geometric Mean Titer; GMTR, Geometric Mean Titer Ratio; SCR, Seroconversion Rate; SPR, Seroprotection Rate. to those with pre immunization HI antibodies titers the adults had HI antibodies titers ≥1:40 for H1N1, 57.6% ≥1:40 (H3N2 p=0.002; influenza Bp =0.001). However, for H3N2, and 88.1% for influenza B viruses; among the there was no significant statistical difference in the SCR elderly, 39.3% had HI antibodies titers ≥1:40 for H1N1 for H1N1 vaccine virus (p=0.075) regardless of the virus, 48.2% for H3N2 virus, and 62.5% for influenza B pre-immunization HI antibodies titers (Table 6). In the virus (Table 5). In adults, SPR >70% was achieved for the elderly, SPR >60%, SCR >30% and GMTR >2.0 were three vaccine viruses, however, SCR >40% and GMTR >2.5 demonstrated for the three vaccine antigens (Table 5). The were described only for the H3N2 vaccine virus (Table 5). elderly with pre-immunization HI antibodies titers <1:40 Adults with pre-immunization HI antibodies titers <1:40 had significantly higher GMTRs for H1N1, H3N2 and had significantly higher GMTRs for the three vaccine for influenza B vaccine viruses when compared to those viruses when compared to those with pre-immunization HI with pre-immunization HI antibodies titers ≥1:40 (H1N1: antibodies titers ≥1:40 (H1N1: p=0.002; H3N2: p<0.001; p=0.005; H3N2: p<0.011; B: p<0.010) (Table 6). The B: p=0.028) (Table 6). SCR for the three vaccine viruses were also statistically The SCR for the three vaccine viruses was also higher among the elderly with pre immunization HI significantly higher among adults with pre-immunization antibodies titers <1:40 when compared to those with pre HI antibodies titers <1:40 when compared to those with pre- immunization HI antibodies titers ≥1:40 (H1N1: p=0.016; immunization HI antibodies titers ≥1:40 (H1N1: p=0.019; H3N2: p=0.001; B: p=0.013) (Table 6). H3N2: p=0.001; B: p=0.046) (Table 6). In the elderly, In the 2015 study, 115 (96.6%) out of 119 participants SPR >60% and GMTR >2.0 were demonstrated for the had pre and post- vaccination blood samples collection, three vaccine antigens, but SCR >30% only for the H3N2 therefore, were included in the immunogenicity analysis: and influenza B viruses (Table 5). The elderly with pre- 59 adults and 56 elderly. Before immunization, 78.0% of immunization HI antibodies titers <1:40 had significantly

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Table 6 - Geometric mean titer ratio, seroprotection and seroconversion rates distributed by age group study year and pre-vaccination hemagglutination-inhibition antibodies titers

Adults Elderly RESULTS <1:40 ≥1:40 p* <1:40 ≥1:40 p* n (CI 95%) n (CI 95%) n (CI 95%) n (CI 95%) 2013 Study GMTR H1N1 21 13.6 (7.9-23.4) 21 2.1 (1.6-2.8) <0.001 6 25.4 (9.4-68.6) 7 3.3 (1.6-6.7) 0.009 H3N2 21 15.0 (8.5-26.5) 21 6.1 (3.8-10.0) 0.023 9 16.0 (5.5-46.4) 4 2.0 (2.0-2.0) 0.011 B 21 5.9 (4.4-8.0) 21 2.2 (1.5-3.3) <0.001 9 5.4 (2.7-11.1) 4 2.8 (0.4-19.1) 0.337 SCR H1N1 21 90.5% (69.6-98.8) 21 23.8% (8.2-47.2) <0.001 6 100% (54.1-100) 7 57.1% (18.4-90.1) 0.192 H3N2 21 85.7% (63.7-97.0) 21 71.4% (47.8-88.7) 0.454 9 88.9% (51.8-99.7) 4 0.0% (0.0-60.2) 0.007 B 21 76.2% (52.8-91.8) 21 28.6% (11.3-52.2) 0.005 9 77.8% (40.0-97.2) 4 50.0% (6.8-93.2) 0.530 2014 Study GMTR H1N1 9 4.3 (2.6-7.1) 48 2.3 (2.0-2.8) 0.011 25 5.6 (3.8-8.2) 33 3.0 (2.2-4.0) 0.005 H3N2 13 9.4 (4.0-22.1) 44 2.0 (1.7-2.3) <0.001 11 10.3 (3.9-26.9) 47 2.2 (1.8-2.7) <0.001 B 7 8.0 (2.5-25.8) 50 1.9 (1.6-2.2) <0.001 15 4.6 (2.7-7.9) 43 2.3 (1.8-3.0) 0.010 SCR H1N1 9 66.7% (29.9-92.5) 48 33.3% (20.4-48.4) 0.075 25 68.0% (46.5-85.1) 33 33.3% (18.0-51.8) 0.016 H3N2 13 69.2% (38.6-90.9) 44 20.5% (9.8-35.3) 0.002 11 81.8% (48.2-97.7) 47 27.7% (15.6-42.6) 0.001 B 7 71.4% (29.0-96.3) 50 10.0% (3.3-21.8) 0.001 15 66.7% (38.4-88.2) 43 27.9% (15.3-43.7) 0.013 2015 Study GMTR H1N1 13 3.8 (2.0-7.3) 46 1.6 (1.4-1.9) 0.002 34 2.9 (2.3-3.7) 22 2.0 (1.6-2.5) 0.044 H3N2 25 10.6 (7.5-14.8) 34 3.4 (2.4-4.8) <0.001 29 8.0 (5.5-11.7) 27 4.7 (3.1-7.0) 0.053 B 7 3.3 (1.4-7.9) 52 1.6 (1.4-1.9) 0.028 21 3.7 (2.5-5.6) 35 2.1 (1.6-2.7) 0.014 SCR H1N1 13 38.5% (13.9-68.4) 46 8.7% (2.4-20.8) 0.019 34 29.4% (15.1-47.5) 22 18.2% (5.2-40.3) 0.529 H3N2 25 96.0% (79.6-99.9) 34 55.9% (37.9-72.8) 0.001 29 72.4% (52.8-87.3) 27 63.0% (42.4-80.6) 0.570 B 7 42.9% (9.9-81.6) 52 9.6% (3.2-21.0) 0.046 21 47.6% (25.7-70.2) 35 34.3% (19.1-52.2) 0.401 Abbreviations: GMTR, Geometric Mean Titer Ratio; SCR, Seroconversion Rate. (*) Mann-Whitney test for GMTR and Fisher's exact test for SCR higher GMTRs only for the H1N1 and influenza B vaccine (inactivated split-virion) in the world and has delivered it to viruses when compared to those with pre-immunization HI the annual influenza vaccination campaign of the National antibodies titers ≥1:40 (p=0.044; p=0.014, respectively) Immunization Program. (Table 6). However, there was no significant statistical From 2013 to 2015, the Brazilian Regulatory Health difference in the GMTR for H3N2 vaccine virus (p=0.053) Authority (Agência Nacional de Vigilância Sanitária regardless of the pre-immunization HI antibodies titers - ANVISA) requested IB to perform post-licensure (Table 6). There was no statistically significant difference prospective cohort studies to evaluate the safety and in the SCR, for the three viruses, when one elderly with immunogenicity of the 2013, 2014, and 2015 seasonal pre-immunization HI antibodies titers <1:40 were compared influenza vaccines in health adults and people over 60 to those with pre-immunization HI antibodies titers ≥1:40 years of age. The sample size for each annual study was (H1N1: p=0.529; H3N2: p=0.570; influenza B: p=0.401) established in accordance with ANVISA and followed (Table 6). the EMA’s recommendations for clinical evaluation of trivalent seasonal influenza vaccine. The number of DISCUSSION participants in the 2013 study was lower compared to the number of participants in the 2014 and 2015 studies. Annual vaccination is recommended by WHO as the However, this did not affect the results because the safety most effective way of preventing seasonal influenza. In and immunogenicity data described for the three years Brazil, the National Immunization Program of the Ministry were similar. This confirms that post-licensure surveillance of Health has carried out annual national influenza vaccine (pharmacovigilance activities) is more appropriate for campaigns since 1999. Currently, IB is one of the main the detection of rare or unexpected safety signals and public producers of seasonal trivalent influenza vaccine effectiveness data of the seasonal influenza vaccines and

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they should be obtained through classic efficacy trials and SCR >40% and GMTR >2.5; in 2014, by SPR >70%; and not be based only on immunogenicity data. in 2015 by SPR >70%. For the elderly, in 2013 and 2014 Among the strategies of the Division of Clinical Trials the vaccine immunogenicity was demonstrated for the three and Pharmacovigilance of IB for conducting clinical vaccine by SPR >60%, SCR >30% and GMTR >2.0; and studies with immunobiological products produced by in 2015 by SPR >60% and GMTR >2.0. the IB is the establishment of partnerships with national Surprisingly, adequate seroconversion frequency values institutions linked to the Unified Health System (SUS), ‌‌were not observed for the three vaccine viruses among such as Brazilian universities and the Adolfo Lutz Institute. adults in the 2014 study and specifically for the H1N1 and These partnerships have made it possible to integrate influenza B viruses in the 2015 study. However, when one basic immunobiological research, vaccine development analyses the frequency of seroconversion in the 2014 study and production, and clinical studies, thus completing the among adults, significantly higher values‌‌ of this frequency innovation process characterized by the availability of were observed for H3N2 and influenza B vaccine viruses vaccines produced by Brazilian public laboratories for SUS. among participants who had pre-immunization HI antibodies The 2013, 2014 and 2015 influenza vaccine studies titers <1:40 compared to those with pre-immunization HI included the participation of the Adolfo Lutz Institute, the antibodies titers ≥1:40. Furthermore, the frequency of CRIE-HCFMUSP and ICR-HCFMUSP. The total number seroconversion ended up being greater than 40% among of participants in the three studies was 299: 60 in 2013; 120 those with pre-immunization HI antibodies titers <1:40. in 2014; and 119 in 2015. The 2013 study was the one with In the 2015 study, significantly higher seroconversion rates the lowest number of participants (47 adults and 13 elderly) were also observed for H1N1 and influenza B vaccine probably due to the late start of the study in the clinical site viruses in adults with pre-immunization HI antibodies when the influenza annual immunization campaign was titers <1:40 compared to those with pre-immunization already close to the end. HI antibodies titers =1: 40. However, a frequency of Overall, the vaccines were well-tolerated with no seroconversion > 40% for the H1N1 vaccine virus among unexpected events or new safety signals. Only in the 2014 adults with pre-immunization HI antibodies titers <1:40 and 2015 studies, the incidence of adverse reactions was antibody was not observed. These findings suggest that, in statistically higher in adults when compared to the elderly. adults, the level of pre-immunization HI antibodies may The adverse reactions observed in adults and elderly, in the impact on the frequency of seroconversion to influenza three studies, were classified as mild or moderate, and no vaccination and that this impact is strain-specific. severe adverse reaction was reported. Besides, they were predominantly observed within 24 h after vaccination and CONCLUSION disappeared up to seven days. These findings have also been demonstrated for other trivalent seasonal influenza vaccines The results of this study demonstrated that the 2013, (split-virion, inactivated)12,13. 2014 and 2015 seasonal influenza vaccines (inactivated The most frequent adverse reactions, in the three studies, split-virion) produced integrally by the IB were overall well- were local pain at the injection site and headache, both in tolerated and safe, since no unexpected events or new safety adults and in the elderly. These adverse reactions have also signs were observed. In addition, these vaccines have been been described in the literature for other trivalent seasonal shown to be immunogenic based on the immunogenicity influenza vaccines (split-virion, inactivated)14-17. Fever criteria defined by the EMA. The satisfactory safety and had a low incidence in the three studies; it is known to be immunogenicity profiles demonstrated over the three study associated with trivalent inactivated influenza vaccines years confirmed the production consistency of the seasonal but is not considered a serious event nor is associated with influenza vaccine (inactivated split-virion) by the IB. complications18. According to EMA’s recommendations7,8, trivalent ACKNOWLEDGMENTS seasonal influenza vaccine (split-virion inactivated) is considered immunogenic when one of the following Our acknowledgments to all the study participants, parameters is demonstrated: a) in adults: SCR >40% the researchers from the clinics, from the laboratory that and/or SPR >70%, and/or HI antibody GMTR >2.5; b) in the performed the assays and the colleagues from the Division elderly: SCR >30% and/or SPR >60% and/or HI antibody of Clinical Trials and Pharmacovigilance from Instituto GMTR >2.0. Butantan. We thank all the institutions involved: Instituto In 2013, the vaccine immunogenicity in adults was Butantan, Fundação Butantan, Instituto Adolfo Lutz, demonstrated for the three vaccine viruses by SPR >70%, CRIE‑HCFMUSP, and ICR-HCFMUSP.

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